1. Field of the Invention
The present invention relates to a microbiological process for preparing sphingolipids, especially, tetraacetylphytosphingosine(TAPS), using a novel yeast
Pichia ciferrii DSCC 7-25 under defined fermentation conditions. Further, this invention concerns a novel yeast Pichia ciferrii DSCC 7-25 and it's isolation method from the parental Pichia ciferrii strain ATCC 14091.
2. Description of Prior Art
The term "sphingolipids" refers to a group of lipids derived from sphingosine. Further, sphingolipids contain sphingosine, dihydrosphingosine or phytosphingosine as a base in amide linkage with a fatty acid. Sphingosine or phytosphingosine bases may be used as starting materials in the synthesis of a particular group of sphingolipids, namely ceramides. Ceramides are main lipid component of the stratum corneum, which has an important barrier function. Therefore, the skin cosmetic products having ceramides has a function for moisture-retaining properties of the skin.
Currently, heterogenous sphingolipid preparations for cosmetics are mainly extracted from animal sources. Obviously, this is a rather costly process on an industrial scale. Moreover, it has been found that these materials are potentially unsafe due, for example, to the possible presence of bovine spongiform encephalomyelitis (BSE) in bovine tissue. Thus, the cosmetic industry has demonstrated an increasing interest in new sources of pure, well-defined sphingolipids, which are obtained from sources other than animal tissues.
Microorganisms such as the yeasts Pichia ciferrii, formerly indicated as Hansenula ciferrii and Endomycopsis ciferrii (Stodola and Wickerham, 1960; Wickerham and Stodola, 1960; Wickerham et al., 1954; Wickerham, 1951) have been found to produce sphingolipids as such, as well as sphingosine, phytosphingosine and/or derivatives thereof. This discovery provides sources for sphingolipids themselves and for starting materials for the production of other commercially valuable compounds which could offer a valuable alternative to the use of animal sources of these compounds.
The biosynthetic pathway of tetraacetylphytosphingosine(TAPS) in Pichia ciferrii was described by Barenholz et al (1973). The biosynthetic pathway for sphingosine and dihydrosphingosine is proposed by Dimari et al. (1971). Barenholz et al. (1971 & 1973) investigated the metabolic background of the production of TAPS and other sphingolipid bases in four strains of Pichia ciferrii. In the later study, the profiles of four microsomal enzymes specific for the biosynthesis of acetylated sphingosine bases of a low (Pichia ciferrii NRRL Y-1031, E-11, sex b, 8-20-57) and a high producer (Pichia ciferrii NRRL Y-1031, F-60-10) were compared. It was found that the specific activity of 3-keto dihydrosphingosine synthetase and the long-chain base acetyl-CoA acetyltransferase were increased 5-10 fold and 30 fold respectively, as compared with the low producer, whereas the activities of palmityl thiokinase and 3-ketodihydrosphingosine reductase were similar. This indicates that in the low producer, the activity of the 3-ketodihydrophingosine synthetase and the long-chain base acetyl-CoA acetyltransferase is the limiting steps in the synthesis of acetylated sphingosines. Under the defined growth conditions, Pichia ciferrii NRRL Y-1031 F-60-10 was found to produce 300 .mu.moles/L sphingosine (about 0.15 g/L) bases, of which, at least 250 .mu.moles/L were extracellular. Even where culture conditions were optimized for TAPS production, only 0.485 g/L TAPS (0.024 g TAPS/g dry yeast) was obtained (Maister et al., 1962).
Maister, using the F-60-10 mating type strain, was able to produce up to 300 mg/L in a pilot scale batch mode fermentation using glucose as a carbon source at 25.degree. C. The TAPS produced is the D-D-erythroisomer, which has the same stereochemistry as the phytosphingosine occurring in the human skin. TAPS may be easily deacetylated to phytosphingosine. However, the yields of TAPS are too low to be of any practical value for commercial production.
Recently, many researchers have been attempted to improve the productivity of sphingolipids using mutant cells of Pichia ciferrii. According to the disclosure in WO 94/10131 (PCT/GB93/02230) maximum 2700 mg/L of TAPS production was reported using Pichia ciferrii NRRL Y-1031 F-60-10 in fed batch mode fermentation with 22.5 mg/L/h of TAPS productivity. Further, in WO 95/12683 (PCI/EP 94/03652), mutants derived from the mating type strain of Pichia ciferrii F-60-10 showed 40.about.60% increased TAPS productivity compared to the parental strain. On the other hand, EP 0 688 871 A2 disclosed the selection and isolation of novel mutant of Pichia ciferrii F-60-10. Using this mutant, average 500.about.1,000 mg/L and maximum 5,000 mg/L of TAPS production was reported, even though the productivity of TAPS is only 30.about.42 mg/L/h.
However, any of the yeast strains studied to date, even Pichia ciferrii NRRL Y-1031 F-60-10, does not produce sufficient amounts of sphingolipid bases such as sphingosine, phytosphingosine or derivatives thereof to be an efficient, economically attractive source of such compounds.
In the early studies of Wickerham and his colleagues(Wickerham and Stodola, 1960), the production of sphingolipids, specifically TAPS was observed to be related to sexuality of the Pichia ciferrii strains. A high TAPS producer mating type NRRL Y-1031, F-60-10 was the one of the mating type isolate derived from the parental strain NRRL Y-1031(ATTC 14091), which was diploid. They also reported that a mating type of one sex(a) had a tentency of producing much higher level of TAPS than the other sex(b). It appeared, however, that there were some other genetic factor(s) affecting production of sphingolipids than sexuality. The strain Y-1031 mating type 11 produced much less TAPS than the strain Y-1031 mating type F-60-10 eventhough it had same sex type with the mating type F-60-10.
Based on the previous findings described above, we reasoned that genetic recombinations during meiosis of a diploid Pichia ciferrii, which results in formation of haploid spores, could give rise to a novel haploid mating type Pichia ciferrii strain with higher TAPS production yield than mating type F-60-10. In addtion, we employed a selection scheme that favors isolation of high producer of TAPS out of the spore pools. Calcium ion has been shown to affect the biosynthesis of sphingolipids by modulating activities of key enzymes involved in the pathway. Depletion of calcium ions by addition of EGTA that chelates calcium ions in the selection medium prevents the yeast cells to grow probably because it prvents synthesis of sphingolipids in the cell. Therefore, new haploid isolates that can grow in those selection environment are likely high producers of TAPS.